1. Field of the Invention
This invention relates to assay for a ligand and materials therein, and, more particularly, relates to an assay including capture of ligand directly on a solid support precoated with a nonspecific agent.
2. Background of the Invention
A variety of assay systems which are both rapid and sensitive has been developed to detect or determine the concentration of a ligand in a liquid. Conventional immunoassays depend on the binding of the ligand to a specific antiligand, and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reagents in labeled form, the labeled reagent often being referred to as the tracer.
Various means for labeling have been developed. Radioimmunoassay (RIA) procedures use radioisotopes as labels, provide high levels of sensitivity and reproducibility, and are amenable to automation for rapid processing of large numbers of samples. Fluoroimmunoassay (FIA) uses fluorochromes as labels, and provides direct detection of the label by exciting the dye with excitation radiation of appropriate wavelength and detecting fluorescence therefrom.
Enzymes have also been used as labels in immunoassay. In enzyme immunoassay (EIA), the enzyme labeled, reagents are cheap to prepare and are highly stable thus giving a long shelf life, yet yield assays which approach the sensitivity of radioimmunoassay and which give objective results that can be determined either visually or with rather simple equipment, such as a spectrophotometer.
In conventional EIA, an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a color which is measured. Often, an unconjugated component, such as an antibody is immobilized on a solid support and serves to capture antigen in a specific binding reaction. Representative of such conventional EIA is U.S. Pat. No. 3,654,090 to Schuurs et al.
Bucher et al. in U.S. Pat. No. 4,588,680 discloses assay for the M protein of various viruses, including Influenza A,B and C viruses. The assay includes disruption of the virus to release the M protein which is absorbed directly onto a polymeric support.
PCT published application WO 86/02733 discloses assay for Herpes simplex virus (HSV) in which antigenic glycoproteins gA/B, gC and gD are absorbed. directly onto a polymeric support or captured by specific monoclonal antibodies on the support.
Sankolli et al. in Journal of Immunological Methods 104, 191 (1987) discloses an immunoassay for estradiol in which a solid support is pretreated with a specific anti IgG antibody. This antibody captures specific antiestradiol antibody which in turn captures estradiol and provides an assay of improved reproducibility.
Armstrong et al. in U.S. Pat. No. 4,497,899 discloses an assay for Chlamydia antigen in which the antigen is absorbed directly onto a solid support such as a bead, tube, strip, disk or microtiter plate. The absorbed antigen is then assayed by any conventional EIA, RIA or FIA technique.
In solid phase immunoassay, particularly for large molecular weight molecules, there is often a tendency for materials in the sample being assayed to attach in a nonspecific manner to the solid support. A particular cause of loss of assay sensitivity or irreproducibility is nonspecific absorption of labeled antiligand conjugate (tracer) directly onto binding sites of the solid support which are not filled by antiligand. This problem has conventionally been addressed by blocking unoccupied binding sites, subsequent to application of antiligand, with a protein which does not react with the labeled conjugate. Cole et al., in U.S. Pat. No. 4,407,943, discloses coating a porous membrane with a water insoluble protein, such a zein, affixing an antigen or antibody to the zein layer, and immobilizing an immunochemically neutral protein on the antigen to prevent nonspecific binding of extraneous protein.
While this conventional post blocking has improved assay sensitivity, the sequential application of antiligand and blocking protein to a solid phase prior to ligand binding is cumbersome, time consuming and wasteful of expensive antiligand. Accordingly, there is a need for further improvement in solid phase immunoassay technology, particularly with respect to avoidance of nonspecific binding.